Recombinant Atractaspis bibroni Bibrotoxin

What is Bibrotoxin and how does it differ structurally from other sarafotoxins?

Structurally, bibrotoxin contains four cysteine residues that form two disulfide bridges, creating a rigid scaffold that is essential for receptor recognition. Like other sarafotoxins, bibrotoxin shares significant sequence similarity with endothelins, though with variations typically at positions 5, 6, 7, and 17 that distinguish the snake venom peptides from mammalian endothelins .

What receptors does Bibrotoxin target and what is known about its binding mechanism?

Bibrotoxin primarily targets endothelin receptors (ETA and ETB), which are class A G protein-coupled receptors (GPCRs). Like other members of the endothelin/sarafotoxin family, bibrotoxin binds to these receptors with high affinity, initiating signal transduction pathways that lead to vasoconstriction and other cardiovascular effects .

The binding mechanism likely follows the general pattern observed with class A GPCRs, where the ligand interacts directly with the transmembrane domain of the receptor. This differs from the two-site/two-step mechanism observed in class B GPCRs, where an initial high-affinity interaction between the ligand's C-terminus and the receptor's N-terminal domain positions the ligand's N-terminus to interact with the receptor's transmembrane region . Current evidence suggests that the entire 21-amino acid sequence of bibrotoxin is involved in receptor recognition and activation, with the specific Lys4Ala substitution potentially modulating its receptor subtype selectivity compared to other sarafotoxins.

What expression systems are most effective for recombinant Bibrotoxin production?

For successful recombinant production of bibrotoxin, bacterial expression systems using modified E. coli strains optimized for disulfide bond formation (such as Origami or SHuffle) represent a viable approach. These strains contain mutations in thioredoxin reductase and glutathione reductase genes, creating an oxidizing cytoplasmic environment that facilitates proper disulfide bond formation.

Alternatively, yeast expression systems (Pichia pastoris or Saccharomyces cerevisiae) may offer advantages for proper folding of disulfide-rich peptides like bibrotoxin. The methodology should include:

• Gene synthesis with codon optimization for the selected expression host

• Fusion with a solubility-enhancing tag (SUMO, thioredoxin, or MBP)

• Inclusion of a precision protease cleavage site between the tag and bibrotoxin sequence

• Controlled, slow induction conditions (lower temperature, reduced inducer concentration)

• Periplasmic targeting in bacterial systems to enhance disulfide bond formation

For researchers requiring post-translational modifications or concerned about endotoxin contamination, mammalian expression systems using CHO or HEK293 cells may be preferable, though at higher production costs and reduced yields compared to microbial systems.

What purification strategies yield the highest purity recombinant Bibrotoxin?

A multi-step purification strategy is essential for obtaining high-purity recombinant bibrotoxin suitable for structural and functional studies:

• Initial capture: Affinity chromatography targeting the fusion tag (IMAC for His-tagged constructs, amylose for MBP fusions)

• Tag removal: Site-specific protease digestion (TEV, PreScission, or SUMO protease) followed by reverse affinity chromatography

• Intermediate purification: Ion-exchange chromatography (typically cation exchange given bibrotoxin's positive charge at physiological pH)

• Polishing step: Size-exclusion chromatography to remove aggregates and ensure monomeric preparation

• Final step: Reversed-phase HPLC to achieve >98% purity required for pharmacological studies

Throughout the purification process, it is critical to maintain reducing agent-free buffers during the final folding steps to allow proper disulfide bridge formation. The native conformation can be verified by circular dichroism spectroscopy and mass spectrometry to confirm the correct disulfide bonding pattern and absence of mixed disulfides.

How does the amino acid substitution (Lys4Ala) affect Bibrotoxin's structure compared to S6b?

The Lys4Ala substitution in bibrotoxin represents a significant change from the positively charged lysine in S6b to the small, hydrophobic alanine residue. This substitution likely affects:

• Electrostatic surface properties: Reduction in positive charge may alter receptor surface interactions

• Conformational dynamics: The smaller alanine side chain potentially increases local flexibility in the N-terminal region

• Hydrophobic interactions: Altered hydrophobicity profile may influence membrane interaction properties

The structural consequences of this substitution can be assessed through comparative molecular dynamics simulations of bibrotoxin and S6b, examining differences in conformational flexibility, solvent accessibility of key residues, and predicted receptor docking orientations . Experimental validation through NMR structural studies would provide definitive evidence of any structural perturbations induced by this substitution.

The position of this substitution is particularly interesting given that it occurs in the N-terminal region of the peptide, which in related endothelin peptides is crucial for receptor activation, suggesting potential functional divergence between bibrotoxin and other sarafotoxins.

What structural techniques provide the most informative data for recombinant Bibrotoxin analysis?

Multiple complementary structural techniques should be employed for comprehensive characterization of recombinant bibrotoxin:

NMR spectroscopy is particularly valuable for bibrotoxin structural studies, as the relatively small size (21 amino acids) makes it ideally suited for solution NMR analysis. Two-dimensional experiments (TOCSY, NOESY, HSQC) can establish intramolecular contacts and determine the three-dimensional structure, while hydrogen-deuterium exchange experiments can assess solvent accessibility and structural rigidity conferred by the disulfide bridges.

What are the comparative binding affinities of Bibrotoxin for ETA versus ETB receptors?

While specific comparative binding data for bibrotoxin against ETA and ETB receptors is limited in the provided search results, methodological approaches to determine these values should include:

• Competitive binding assays using radiolabeled endothelin-1 or sarafotoxin S6b

• Surface plasmon resonance (SPR) with immobilized receptor ectodomains

• Time-resolved fluorescence resonance energy transfer (TR-FRET) assays

• Cellular calcium mobilization assays in cells expressing either ETA or ETB

For quantitative analysis, researchers should calculate Ki values using the Cheng-Prusoff equation from IC50 values obtained in competitive binding assays, ensuring equilibrium conditions are maintained throughout the experiments by using appropriate incubation times.

How does Bibrotoxin affect cellular signaling pathways compared to endogenous endothelins?

Bibrotoxin, through its interaction with endothelin receptors, likely activates similar signaling pathways as endogenous endothelins, including:

• Gq/11-coupled pathways: Activation of phospholipase C, IP3 generation, and intracellular calcium mobilization

• G12/13-coupled pathways: RhoA activation leading to cytoskeletal reorganization

• β-arrestin recruitment: Receptor internalization and potential signaling through MAP kinase cascades

To characterize these pathways experimentally, researchers should employ:

• FRET-based calcium sensors for real-time monitoring of intracellular calcium dynamics

• Phospho-specific antibodies to detect ERK1/2, p38, and JNK activation

• RhoA activation assays using rhotekin pull-down approaches

• β-arrestin recruitment assays using enzyme complementation or BRET technologies

The key question for bibrotoxin research is whether the Lys4Ala substitution alters signaling bias compared to other sarafotoxins or endothelins, potentially favoring certain pathways over others. This can be assessed through systematic quantification of multiple signaling outputs and construction of bias plots comparing equiactive concentrations across different pathways.

How can recombinant Bibrotoxin be used as a tool to study endothelin receptor pharmacology?

Recombinant bibrotoxin represents a valuable tool for endothelin receptor research due to its structural similarity to endogenous ligands but with distinct features that can provide insights into receptor-ligand interactions:

• Structure-activity relationship studies: Systematic mutagenesis of bibrotoxin can reveal essential residues for binding and activation of endothelin receptors. Creating a library of bibrotoxin variants with single amino acid substitutions allows mapping of the pharmacophore responsible for receptor subtype selectivity .

• Receptor binding site mapping: Photoaffinity labeling with derivatized bibrotoxin containing photoreactive groups can identify specific contact points within the receptor binding pocket.

• Allosteric modulator discovery: Bibrotoxin can serve as a probe ligand in high-throughput screening assays to identify compounds that modulate endothelin receptor function through allosteric mechanisms.

• Biased signaling investigation: Comparing signaling profiles of bibrotoxin with other endothelin family members can reveal pathway-selective agonism, informing the development of biased ligands with therapeutic potential.

The unique Lys4Ala substitution in bibrotoxin provides a natural probe for understanding how changes in the N-terminal region affect receptor recognition and activation, potentially guiding the design of selective endothelin receptor modulators.

What insights can comparative studies between Bibrotoxin and long-sarafotoxins provide?

Comparative studies between bibrotoxin and long-sarafotoxins (l-SRTXs) offer unique opportunities to understand the functional significance of C-terminal extensions in this peptide family:

Recent transcriptomic studies revealed that some Atractaspis species produce hybrid toxins combining bibrotoxin-like sequences with C-terminal extensions. Specifically, a full-length mature SRTX sequence from Atractaspis aterrima combines the 21 first amino acid residues attributed to bibrotoxin with the C-terminal extension "DEP" found in long-SRTXs of Atractaspis microlepidota .

Comparative functional studies should examine:

• Receptor binding kinetics: The C-terminal extension may affect association/dissociation rates

• Receptor subtype selectivity: Altered ETA/ETB preference compared to standard bibrotoxin

• Signaling profiles: Potential differences in G-protein coupling or β-arrestin recruitment

• Physiological effects: Differences in vasoconstriction potency, duration of action, and tissue specificity

Understanding these differences would provide insights into the natural evolution of these toxins and how structural modifications alter their pharmacological properties. This knowledge could guide the rational design of modified peptides with tailored pharmacological profiles for research and potential therapeutic applications.

What are optimal protocols for assessing Bibrotoxin receptor binding kinetics?

For rigorous characterization of bibrotoxin-receptor interactions, researchers should implement multiple complementary approaches:

• Real-time binding kinetics using SPR or BLI:

  • Immobilize purified receptor (or receptor-enriched membranes) on sensor surface

  • Flow bibrotoxin at multiple concentrations (0.1-100× estimated KD)

  • Determine association (kon) and dissociation (koff) rate constants

  • Calculate KD from ratio koff/kon

  • Control experiments should include known endothelin receptor ligands

• Equilibrium binding using radioligand displacement:

  • Use [125I]-ET-1 as tracer ligand

  • Perform homologous and heterologous competition experiments

  • Incubate for sufficient time to reach equilibrium (typically 2-4 hours at 4°C)

  • Separate bound from free ligand via filtration

  • Analyze data using one- and two-site binding models

• Residence time determination:

  • Pre-equilibrate receptors with bibrotoxin

  • Initiate dissociation with excess unlabeled competitor

  • Sample at multiple time points to construct dissociation curves

  • Calculate receptor-ligand complex half-life (t1/2)

When designing these experiments, researchers should account for potential receptor internalization during longer incubations and consider the use of receptor mutants to probe specific binding interactions hypothesized to be important based on structural models.

How should cell-based functional assays be designed to evaluate recombinant Bibrotoxin activity?

Rigorous cell-based assays are essential for functional characterization of recombinant bibrotoxin:

• Calcium mobilization assays:

  • Use cells stably expressing either ETA or ETB receptors

  • Load cells with fluorescent calcium indicators (Fluo-4 AM or Fura-2 AM)

  • Measure real-time calcium responses to increasing bibrotoxin concentrations

  • Include ET-1 and known sarafotoxins as reference compounds

  • Calculate EC50 values and maximum responses for comparison

• MAP kinase activation:

  • Treat receptor-expressing cells with bibrotoxin for varying durations (5-60 min)

  • Lyse cells and analyze by western blot for phosphorylated ERK1/2

  • Quantify dose-dependency and time-course of activation

  • Compare with endogenous endothelins for signal magnitude and duration

• Vasoconstriction assays:

  • Use isolated vessel preparations (e.g., rat aortic rings)

  • Measure isometric tension in response to cumulative bibrotoxin concentrations

  • Test in presence of selective ETA and ETB antagonists to determine receptor contribution

  • Compare potency and efficacy with ET-1 and other sarafotoxins

A critical experimental design consideration is the preparation of recombinant bibrotoxin stocks of verified purity and concentration, with confirmation of proper disulfide bond formation, to ensure consistent and reproducible results across different assay platforms.

What evolutionary insights can be gained from studying Bibrotoxin's relationship to other snake venom peptides?

Bibrotoxin represents an important evolutionary node in the diversification of snake venom peptides within the Atractaspididae family. Molecular evolution studies provide several key insights:

Recent transcriptomic analysis of Atractaspis venoms has revealed unexpected diversity in the toxin arsenal. While sarafotoxins like bibrotoxin were previously thought to be the predominant toxins, high-throughput transcriptomic approaches have uncovered a surprising variety of other well-characterized snake venom components, including three-finger toxins (3FTxs) .

The 3FTxs from Atractaspis aterrima show evidence of rapid evolution under the influence of positive selection. Site-model 8 computed an ω value of 1.75 for these toxins, with Bayesian Empirical Bayes approach identifying 17 positively selected sites (39% of total sites) . This suggests that these toxins are undergoing adaptive evolution, possibly in response to prey resistance mechanisms or shifts in prey types.

Comparative analysis of bibrotoxin with other sarafotoxins and endothelins can illuminate the selective pressures that have shaped these peptides. The specific Lys4Ala substitution that distinguishes bibrotoxin from S6b may represent a key evolutionary adaptation that potentially alters receptor subtype selectivity or resistance to prey defensive mechanisms.

What structure-function analyses have identified key functional residues in Bibrotoxin?

Structure-function analyses of bibrotoxin and related peptides have highlighted several key features:

• The four cysteine residues forming two disulfide bridges (Cys1-Cys15 and Cys3-Cys11) are absolutely conserved among all endothelins and sarafotoxins, creating the essential structural scaffold required for receptor recognition .

• The C-terminal region (residues 8-21) is highly conserved across the endothelin/sarafotoxin family and is primarily responsible for high-affinity binding to receptors, while the N-terminal region (residues 1-7) shows greater variability and contributes to receptor subtype selectivity and activation .

• Position 4, where bibrotoxin contains an alanine instead of the lysine found in S6b, likely plays a role in modulating receptor interactions and possibly alters the activation mechanism or signaling bias .

To fully characterize structure-function relationships, systematic alanine scanning mutagenesis of recombinant bibrotoxin would be valuable. By replacing each non-alanine residue with alanine and assessing the impact on receptor binding and activation, researchers can map the contribution of each amino acid to bibrotoxin's pharmacological properties.

Additionally, chimeric peptides combining regions of bibrotoxin with corresponding regions of endothelins or other sarafotoxins can help delineate which structural elements confer specific functional properties, potentially guiding the design of peptides with tailored pharmacological profiles.

What are the main technical challenges in working with recombinant Bibrotoxin and how can they be addressed?

Researchers working with recombinant bibrotoxin face several technical challenges that require specific methodological approaches:

• Correct disulfide bond formation: The presence of two disulfide bridges necessitates careful oxidative folding conditions. Implementation of controlled oxidation using glutathione redox buffers (typically 1:10 ratio of reduced:oxidized glutathione) can promote correct disulfide pairing. Alternatively, directed disulfide formation using orthogonal protection strategies during solid-phase peptide synthesis offers precise control over disulfide connectivity.

• Solubility issues: Small, highly structured peptides like bibrotoxin may exhibit aggregation tendencies. Addition of solubilizing agents (0.1% Tween-20 or 1 mM CHAPS) to storage buffers and experimental solutions can mitigate aggregation. Long-term storage should utilize lyophilized aliquots reconstituted immediately before use.

• Quantification accuracy: Accurate concentration determination is critical for binding and functional studies. Amino acid analysis provides the most reliable absolute quantification, while accounting for the absence of aromatic residues (Trp, Tyr) which limits accuracy of spectrophotometric methods.

• Receptor expression systems: Endothelin receptors can be challenging to express at levels required for binding studies. Utilization of expression systems with inducible promoters, combined with receptor-GFP fusion constructs for monitoring expression levels, can optimize experimental conditions.

Researchers should implement rigorous quality control procedures, including analytical HPLC, mass spectrometry, and circular dichroism, to verify batch-to-batch consistency before conducting detailed pharmacological characterization.

What are promising future research directions involving recombinant Bibrotoxin?

Several promising research directions could significantly advance understanding of bibrotoxin and expand its research applications:

• Cryo-EM structural studies: Recent advances in cryo-electron microscopy now enable structural determination of GPCRs in complex with their ligands. A structure of bibrotoxin bound to endothelin receptors would provide unprecedented insight into the molecular details of this interaction and guide structure-based drug design efforts targeting these receptors.

• Bibrotoxin-based molecular probes: Development of fluorescently labeled bibrotoxin derivatives could provide valuable tools for studying receptor localization, trafficking, and dynamics in live cells. Strategic placement of fluorophores at positions that don't interfere with receptor binding would be essential for maintaining native pharmacology.

• Peptide engineering approaches: Using bibrotoxin as a scaffold, researchers could engineer modified peptides with enhanced stability, altered receptor subtype selectivity, or biased signaling properties. Incorporation of non-natural amino acids through amber codon suppression technologies offers exciting possibilities for expanding the chemical diversity beyond the standard 20 amino acids.

• Therapeutic potential exploration: While bibrotoxin itself is a toxin, engineered derivatives might have therapeutic applications in conditions where modulation of endothelin receptor signaling is beneficial. Antagonistic variants could potentially address conditions characterized by endothelin system overactivation, such as pulmonary arterial hypertension.

• Broader transcriptomic exploration: Recent studies have revealed unexpected diversity in Atractaspis venoms . Comprehensive transcriptomic and proteomic profiling across multiple Atractaspis species could reveal novel bibrotoxin variants with unique pharmacological properties worth investigating.

These research directions would benefit from interdisciplinary collaboration between structural biologists, pharmacologists, peptide chemists, and translational researchers to fully realize the potential of bibrotoxin as both a research tool and therapeutic lead.